TLF in Spermiogenesis 947

Development 129, 945-955 (2002) 945 Printed in Great Britain © The Company of Biologists Limited 2002 DEV2828 Distinct functions of TBP and TLF/TRF2 during spermatogenesis: requirement of TLF for heterochromatic chromocenter formation in haploid round spermatids Igor Martianov1,†, Stefano Brancorsini1,†, Anne Gansmuller1, Martti Parvinen2, Irwin Davidson1,‡ and Paolo Sassone-Corsi1,‡ 1Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, B.P. 163, 67404 Illkirch Cédex, C.U. de Strasbourg, France 2Department of Anatomy, University of Turku, 20520 Turku, Finland †These authors contributed equally to this work ‡Authors for correspondence Accepted 23 November 2001 SUMMARY TLF (TBP-like factor) is a protein commonly thought to pachytene spermatocytes, suggesting a function prior to the belong to the general transcription initiation complex. TLF apoptosis of the haploid cells. A refined study of TLF- is evolutionarily conserved and has been shown to be deficient mice reveals defective acrosome formation in early essential for early development in C. elegans, zebrafish and stage spermatids. Most importantly, our results uncover an Xenopus. In mammals however, TLF has a specialised unsuspected function of TLF in chromatin organisation. function, as revealed by targeted mutation of the gene in Indeed, early spermatids in TLF-deficient mice display a the mouse germline. The TLF mutation elicits a complete fragmentation of the chromocenter, a condensed structure arrest of late spermiogenesis and increased haploid cell formed by the association of centromeric heterochromatin apoptosis. We explored in more detail the molecular and containing the HP1 proteins. This defect is likely to be function that TLF plays in the differentiation program of the primary cause of spermatogenic failure in the TLF male germ cells. A comparison of TBP and TLF reveals mutant mice. drastic differences, both in their temporal expression pattern and in their intracellular location. While TBP is ubiquitously expressed, TLF expression is strictly Key words: Spermatogenesis, TLF, TBP, Chromatin, Chromocenter, developmentally regulated, being very high in late HP1, Mouse. Rat INTRODUCTION Burley, 1994; Kim et al., 1993) which is responsible for DNA binding, and interaction with TFIIA, TFIIB and TAFs (Liu et RNA polymerase II transcription initiation in eukaryotes al., 1998; Nikolov et al., 1995; Tan et al., 1996). Two proteins requires the formation of a multiprotein complex around the with strong sequence similarity to the core domain of TBP have mRNA start site. (Hampsey, 1998; Hampsey and Reinberg, been described. The first, Drosophila melanogaster TRF1, is 1999; Orphanides et al., 1996). A central factor in this process expressed in a tissue-specific fashion and functions as both a is transcription factor TFIID comprising the TATA binding RNA polymerase II and polymerase III transcription factor protein (TBP) and 14 additional TBP-associated factors (Hansen et al., 1997; Holmes and Tjian, 2000; Takada et al., (TAFIIs). TFIID plays an important role in transcriptional 2000). At present the gene for this has been described only in regulation through contributing to promoter recognition and Drosophila. acting as a target for transcriptional activators (for reviews, see A second protein with high homology to the TBP core Albright and Tjian, 2000; Bell and Tora, 1999; Gangloff et al., domain has received various names; TLF, TRF2, TLP and TRP 2001; Green, 2000). TBP itself is thought to play a role in (Maldonado, 1999; Moore et al., 1999; Ohbayashi et al., 1999; transcription initiation by all three RNA polymerases through Rabenstein et al., 1999; Teichmann et al., 1999; Wieczorek et its presence in TFIID for polymerase II, SL1 for polymerase I al., 1998). The TLF (TBP-like factor) gene is found in most and TFIIIB for polymerase III (Chiang et al., 1993; Comai et metazoans (Dantonel et al., 1999) and multiple sequence al., 1992; Hernandez, 1993). alignments show that TLFs contain two direct repeats, which TBP has a bipartite structure with a variable N-terminal are well conserved between all TBP-type factors. Despite this domain and a highly conserved C-terminal core domain. The sequence similarity, TLF does not bind classical TATA core domain of TBP folds into a saddle-like structure (Kim and elements (Dantonel et al., 1999; Moore et al., 1999), but 946 I. Martianov and others does interact with TFIIA and TFIIB. Transfection, and while in TLF–/– spermatids the single center is fragmented into overexpression studies in cells and in in vitro experiments, multiple structures. These results show that TLF plays an have shown that TLF associates stably with TFIIA and can act essential role in proper chromatin organisation in developing as a repressor or activator of RNA polymerase II transcription male germ cells. Disorganisation of chromatin domains in the (Moore et al., 1999; Ohbayashi et al., 2001; Teichmann et al., early round spermatids could lead to defects in gene expression 1999). and chromosomal packaging in the elongated spermatids and The physiological function of TLF was first addressed using therefore be the primary cause of differentiation arrest and RNAi in Caenorhabditis elegans where TLF appears to apoptosis of TLF–/– spermatids. function as an essential RNA polymerase II transcription factor (Dantonel et al., 2000; Kaltenbach et al., 2000). Injected embryos die at an early stage of embryogenesis before the MATERIALS AND METHODS onset of morphogenesis. Similar results have been obtained using antisense strategies in Xenopus laevis (Veenstra et al., RNA analysis 2000) and in the zebrafish Danio rerio (Muller et al., 2001). Total RNA was extracted from mouse testis and rat spermatogenic These studies underscored a critical role of TLF in the cells as previously described (Chomczynski et al., 1986) and analyzed embryogenesis of these species. It has been suggested that TLF by RNase protection (Foulkes et al., 1991). Rat spermatocyte and may act as a surrogate for TBP at specific promoters whose spermatid cells were separated by centrifugation as described (Meistrich et al., 1981). The TLF and TBP cDNAs were used to expression is required at the onset of zygotic transcription in [α 32 ] the developing embryos. However, as it has been demonstrated generate a - P UTP antisense riboprobe using an in vitro transcription kit (Promega). CREM riboprobe was as described recently, the situation is radically different in mammals. previously (Molina et al., 1993). In all RNase protection analyses, Indeed, using homologous recombination, it has been shown transfer RNA was used as a control for nonspecific protection. A that mouse TLF is not required for embryogenesis, but that it mouse β-actin riboprobe was used as internal control to monitor the is essential for spermatogenesis (Martianov et al., 2001; Zhang loading of equal amounts of RNA. et al., 2001b). Spermatogenesis is a cyclic process in which diploid Immunoblots and immunohistochemistry spermatogonia differentiate into mature haploid spermatozoa. Rat seminiferous tubule segments at defined stages were isolated Spermatogonia committed for differentiation give rise to using the transillumination-assisted microdissection method spermatocytes which undergo two meiotic divisions, to (Parvinen and Hecht, 1981) and pooled to obtain 4-5 cm, equivalent generate haploid round spermatids. During the process of to 4-5 mg of wet weight of tissue. Protein extracts were made by shearing the tubule segments in 2× boiling Laemmli buffer containing spermiogenesis the haploid round spermatids undergo an 10 mM β-mercaptoethanol. The extracts were analyzed by SDS- elongation phase in which they are sculptured into the shape PAGE and staining with Coomassie Blue stain to normalise each of mature spermatozoa. This entails a major biochemical and preparation. Immunoblotting and chemiluminescence detection were morphological restructuring of the germ cell in which the performed by standard methods. Immunohistochemistry was majority of the somatic histones are replaced by protamines to performed on fixed sectioned seminiferous tubules or from squash pack the DNA into the sperm cell nucleus. In the absence of preparations of microdissected tubules from wild-type or TLF mutant TLF, round step 7 haploid spermatids die through apoptosis as mice as previously described (Martianov et al., 2001; Nantel et al., the transition to elongated spermatids begins. Rare elongating 1996). Antibodies against TBP, TLF and CREM are as previously described (Brou et al., 1993; Delmas et al., 1993; Martianov et al., spermatids remain beyond this stage but rapidly degenerate α such that no spermatids survive beyond step 13. Hence, TLF 2001). The anti-HP1 , 2HP1-1H5 and anti-fibrillarin antibodies are also as described previously (Fomproix et al., 1998; Nielsen et al., is essential for the differentiation of male germ cells. These 2001). results highlighted the non-redundant function of TBP and TLF in mammals and solicited further investigations aimed at identifying their distinct roles. To gain further insight into the molecular function that TLF RESULTS plays in the differentiation program of male germ cells, we have examined in detail the pattern of TLF expression during Developmental and stage-specific expression of TLF spermatogenesis and compared it with that of TBP. Our results and TBP show marked differences in temporal regulation of TBP and To compare the functions of TBP and TLF in developing male TLF as well as differences in their

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